The long-term objectives of this research program are to develop novel and efficient ways to discover small-molecule antagonists of gene expression. In the work proposed herein, we plan to integrate structural biology and combinatorial chemistry to generate inhibitors of HIV reverse transcriptase (RT). We have recently discovered that it is possible to prepare catalytically relevant complexes of RT stalled on a primer: template through disulfide crosslinking of the protein and DNA. We have obtained co-crystals of this complex that diffract to spacings of approximately 3 A. In the proposed studies, we will optimize the conditions for production of high-quality co-crystals of RT complexes, and will prepare additional complexes with DNA:RNA templates, nucleoside inhibitor drugs such as AZT, and drug-resistant mutants of RT. The second major aspect of the proposed studies will involve exploring new methods of biasing combinatorial libraries so as to focus them on a particular surface of a protein or DNA. One method, known as thiol bashing, will employ transient disulfide bond formation to recruit a combinatorial library to an active site region consisting of three clustered aspartase residues, and another method will employ hydrogen-bonding and electrostatic interactions to bias the interaction with the tri-Asp motif. If successful, the proposed studies will not only produce new small- molecule leads for development as anti-HIV agents, but will also provide a new and potentially general strategy for exerting control over gene expression in cells.